Medicaments

ABSTRACT

Methods of prevention or treatment of renal diseases or conditions associated with abnormal ion flux, in particular autosomal dominant polycystic kidney disease, with a modulator of human peroxisome proliferator activated receptor gamma.

FIELD OF THE INVENTION

The present invention is concerned with methods of treatment,pharmaceutical compositions and medicaments for use in treatment. Moreparticularly, the invention relates to modulators of PeroxisomeProliferator Activated Receptor gamma (PPARgamma). In another aspect,the present invention relates to methods for prevention or treatment ofrenal diseases or conditions associated with abnormal ion flux includingautosomal dominant polycystic kidney disease (ADPKD).

BACKGROUND TO THE INVENTION

Peroxisome Proliferator Activated Receptors (PPARs) are orphan receptorsbelonging to the steroid/retinoid receptor superfamily ofligand-activated transcription factors. See, for example Willson T. M.and Whali, W., Curr. Opin. Chem. Biol., 1, pp 235-241 (1997) and WillsonT. M. et. Al., J. Med. Chem., 43, P527-549 (2000). The binding ofagonist ligands to the receptor results in changes in the expressionlevel of mRNAs encoded by PPAR target genes.

Three mammalian Peroxisome Proliferator-Activated Receptors have beenisolated and termed PPAR-alpha, PPAR-gamma, and PPAR-delta (also knownas NUC1 or PPAR-beta). These PPARs regulate expression of target genesby binding to DNA sequence elements, termed PPAR response elements(PPRE). To date, PPRE's have been identified in the enhancers of anumber of genes encoding proteins that regulate lipid metabolismsuggesting that PPARs play a pivotal role in the adipogenic signallingcascade and lipid homeostasis (H. Keller and W. Wahli, TrendsEndocrinol. Metab. 291-296, 4 (1993)).

It has been reported that the thiazolidinedione class of compoundsincluding rosiglitazone and pioglitazone are potent and selectiveactivators of PPAR-gamma and bind directly to the PPAR-gamma receptor(J. M. Lehmann et al., J. Biol. Chem. 12953-12956, 270 (1995)),providing evidence that PPAR-gamma is a possible target for thetherapeutic actions of the thiazolidinediones. Since this observation,activation of this nuclear hormone receptor has been shown to havepleiotropic metabolic and nonhypoglycemic effects. Clinical use of theagents in the treatment of Type 2 diabetes mellitus (or non insulindependent diabetes mellitus (NIDDM)) is associated with sensitization tothe glucose lowering effects of insulin as well as potentiation of otherbiological actions of insulin in target tissues. When used asmonotherapy, there are reports of fluid retention resulting in volumeexpansion and, in some patients, clinical edema. The incidence of edemaappears to be increased when both these agents are used in combinationwith insulin (Nesto R. W. et al, 2003, Circulation, 108, 2941-2948).However, the mechanisms involved in these effects have not been welldescribed but the nature of the presentation suggests an integratedphysiological response which includes an effect on renal salt and waterbalance. PPAR gamma receptors have been found in the renal collectingduct (Guan Y. et al; 2001, Kidney Int. 60, 14-30) and, therefore, thePPAR gamma agonists might be involved directly in renal tubularmetabolism or could have secondary effects on salt and waterhomeostasis.

Autosomal-dominant polycystic kidney disease (ADPKD) is one of the mostprevalent single gene disorders to affect humans with an incidence ofapproximately 1 in 1000 live births in all ethnic groups (Gabow P. A.,1993, N. Engl. J. Med. 329:332-342). The disease is caused by mutationsin the polycystin proteins that initiate a cascade of events resultingin the formation of multiple fluid-filled epithelial cysts whichprogressively destroys the architecture of the kidney leading to severerenal failure. Currently, no therapies exist for ADPKD which account for8-10% of patients requiring kidney transplantation or dialysis (Gabow P.A., 1993, N. Engl. J. Med. 329:332-342). It will therefore beappreciated that it is desirable to identify and develop treatments forthis disease.

The development and growth of ADPKD cysts involve the proliferation ofimmature epithelial cells, changes in the extracellular matrix and theaccumulation of fluid in the cyst cavity. This is driven by CAMPstimulated cell proliferation and Cl-secretion via the cystic fibrosistransmembrane conductance regulator (CFTR) Cl-channel. Thus it isthought that inhibitors at the CF TR Cl-channel may retard cyst growthprincipally by blocking fluid accumulation within the cyst lumen.(Hongyu Li et al., 2004, Kidney International 66; 1926-1938).

The present inventors have found that modulators of the peroxisomeproliferator activated receptor gamma (PPAR gamma) may inhibit anionsecretion via CFTR in renal cells and thus are of potential therapeuticbenefit in the treatment of renal diseases or conditions associated withabnormal ion flux, including ADPKD.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound which is a modulator ofhuman PPAR gamma (hPPAR gamma) or a pharmaceutically acceptable salt orsolvate thereof for use in the treatment of renal diseases or conditionsassociated with abnormal ion flux.

In a further aspect there is provided a pharmaceutical compositioncomprising a hPPAR gamma modulator or a pharmaceutically acceptable saltor solvate thereof for use in the treatment of renal diseases orconditions associated with abnormal ion flux.

In a further aspect the invention provides a method of treating renaldiseases or conditions associated with abnormal ion flux comprisingadministering hPPAR gamma modulator or a pharmaceutically acceptablesalt or solvate thereof.

In a further aspect, the invention provides the use of a hPPAR gammamodulator or a pharmaceutically acceptable salt or solvate thereof inthe manufacture of a medicament for treating renal diseases orconditions associated with abnormal ion flux.

DESCRIPTION OF THE FIGURES

FIG. 1:

Ion transport response of MDCK-C7 cells (short circuit current) tostimulation by ADH in control and amiloride-pretreated cells. The entiretime-course is shown in FIG. 1A and the first 10 minutes shown in FIG.1B.

FIG. 2:

Ion transport response of MDCK-C7 cells to stimulation by ADH in cellspretreated with inhibitors of CFTR. Ion transport is measured asshort-circuit current (SCC). A positive deflection indicates cationmovement from apical to serosal bathing medium or anion movement fromserosal to apical medium.

FIG. 3:

Time course of the ion transport response of MDCK-C7 cells tostimulation by ADH in cells pre-treated for 18 hours with GI262570X. Iontransport is measured as short-circuit current (SCC). A positivedeflection indicates cation movement from apical to serosal bathingmedium or anion movement from serosal to apical medium.

FIG. 4:

Ion transport response of MDCK-C7 cells to stimulation by ADH in cellspretreated for 18 hours with GI262570X. Ion transport is measured asshort-circuit current (SCC). The data are shown as the initial 4 minutesof the response. A positive deflection indicates cation movement fromapical to serosal bathing medium or anion movement from serosal toapical medium.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the term “hPPAR gamma modulator” is meant a compoundwhich alters the activity of the hPPAR gamma receptor either directly(by binding thereto and altering activity) or indirectly (for example,by causing some change in receptor expression and/or degradation rate)thus increasing or decreasing the transcription of PPAR-responsive genesrelative to the PPAR receptor in a basal state—either unliganed in an invitro setting or in an in vitro setting, in the presence of “normal”amounts of endogenous PPAR ligands. This definition thus embracesagonists, partial agonists, antagonists, partial antagonists.

The hPPAR gamma agonists of formula (I) may be agonists or partialagonists of only gamma (“selective agonists”), agonists or partialagonists for two PPAR subtypes including gamma (“dual agonists”), oragonists or partial agonists for all three subtypes (“pan agonists”).

In one aspect the hPPAR gamma modulators are human (h) PPAR gammaagonists. In a further aspect they are also agonists of at least one ofhPPARα or hPPARδ (dual agonists). In one aspect they are pan hPPARagonists.

In a further aspect, the hPPAR gamma modulators are hPPAR gamma agonistsor partial agonists.

As used herein, by “agonists”, or “activating compound”, or “activator”,or the like, is meant those compounds which have a pKi of at least 5.0preferably at least 6.0 to the relevant PPAR, for example hPPAR gamma inthe binding assay described for example in WO 02/059098 (or similarassays), and which achieve at greater than 70% activation of therelevant PPAR relative to the appropriate indicated positive control inthe transfection assay described in WO02/059098 (or similar assays) atconcentrations of 10⁻⁵M or less. More preferably, the compounds of thisinvention achieve greater than 70% activation of at least on human PPARin the relevant transfection assay at concentrations of 1⁻⁵M or less.More preferably the compounds of the invention achieve greater than 70%activation of at least on human PPAR in the relevant transfection assayat concentrations of 1⁻⁷M or less.

Partial agonists can be defined as compounds that transactivate therelevant hPPAR, for example hPPAR alpha in CV1 cells with less than 70%,typically less than 50% fold activation compared to the reference PPARfull agonist in the transfection assays of the type described in WO97/31907. A compound having activity at more than on hPPAR mayindependently demonstrate full or partial agonist activity at eachhPPAR.

The term “treatment” as used herein includes prophylaxis as well asalleviation of established diseases or conditions. The PPAR gammamodulators may be used as compounds or salts or solvates thereof.

In one aspect the renal disease or condition associated with abnormalion flux is ADPKD.

In one aspect the PPAR gamma modulator is described in European Patent306228. This describes a class of PPAR gamma agonists which arethiazolidinedione derivatives for use as insulin sensitisers in thetreatment of Type 2 diabetes mellitus. These compounds haveanti-hyperglycaemic activity. One preferred compound described thereinis known by the chemical name5-[4-[2-(N-methyl-N-(2-pyridyl)amino)ethoxy]benzyl]thiazolidine-2,4-dioneand has been given the generic name rosiglitazone. Salts of thiscompound including the maleate salt are described in WO94/05659.European Patent Applications, Publication Numbers: 0008203, 0139421,0032128, 0428312, 0489663, 0155845, 0257781, 0208420, 0177353, 0319189,0332331, 0332332, 0528734, 0508740; International Patent Application,Publication Numbers 92/18501, 93/02079, 93/22445 and U.S. Pat. Nos.5,104,888 and 5,478,852, also disclose certain thiazolidinedione insulinsensitisers. Specific compounds that may be mentioned include5-[4-[2-(5-ethyl-2-pyridyl)ethoxy]benzyl]thiazolidine-2,4-dione (alsoknown as pioglitazone),5-[4-[(1methylcyclohexyl)methoxy]benzyl]thiazolidine-2,4-dione (alsoknown as ciglitazone),5[[4-[(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)methoxy]phenyl]methyl]-2,4-thiazolidinedione(also known as troglitazone) and5-[(2-benzyl-2,3-dihydrobenzopyran)-5-ylmethyl)thiazolidine-2,4-dione(also known as englitazone).

U.S. Pat. No. 6,294,580 (the disclosure of which is herein incorporatedby reference in its entirety) describes a series of PPAR gamma agonistcompounds not of the thiazolidinedione class but which are instead O-and N-substituted derivatives of tyrosine which nevertheless areeffective as insulin sensitisers in the treatment of Type 2 diabetesmellitus. One such compound has chemical nameN-(2-benzoylphenyl)-O-[2-(5-methyl-2-phenyl-4-oxazolyl)ethyl]-L-tyrosine(also known as2(S)-(2-benzoyl-phenylamino}-3-{4-[2-5-methyl-2-phenyl-oxazol-4-yl)-ethoxy]phenyl}-propionicacid, or by the generic name farglitazar, or GI262570. A method ofsynthesising farglitazar is shown in Example 29.

Further compounds are described in WO 02/062774, WO 02/30895, WO00/08002, WO 02/059098, WO 03/074495.

In a preferred embodiment the compound is farglitazar (particularly thesodium salt), rosiglitazone (particularly the maleate salt),2-({4-[({4-({4-[4-(ethyloxy)phenyl]-1-piperazinyl}methyl)-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)thio]phenyl}oxy)-2-methylpropanoicacid:

(for preparation of this compound, see WO 02/059098),({2-ethyl-4-[({4-({4-[4-(methyloxy)phenyl]-1-piperazinyl}methyl)-2-[4-(trifluoromethyl)phenyl]-1,3-thiazol-5-yl}methyl)thio]phenyl}oxy)aceticacid:

(for preparation of this compound, see WO 02/059098),2-{4-[{2-[2-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-1,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}-2-methylpropanoicacid

(for preparation of this compound, see WO 02/062774).

Where stereocenters exist in compounds which are PPAR modulators, thepresent invention includes the use of all possible stereoisomers andgeometric isomers of such compounds and includes not only racemiccompounds but also the optically active isomers as well. When a compoundis desired as a single enantiomer, it may be obtained either byresolution of the final product or by stereospecific synthesis fromeither isomerically pure starting material or any convenientintermediate. Resolution of the final product, an intermediate or astarting material may be affected by any suitable method known in theart. See, for example, Stereochemistry of Carbon Compounds by E. L.Eliel (Mcgraw Hill, 1962) and Tables of Resolving Agents by S. H. Wilen.Additionally, in situations where tautomers of the compounds arepossible, the present invention is intended to include the use of alltautomeric forms of the compounds.

As used herein, the term “effective amount” means that amount of a drugor pharmaceutical agent that will elicit the biological or medicalresponse of a tissue, system, animal or human that is being sought, forinstance, by a researcher or clinician. Furthermore, the term“therapeutically effective amount” means any amount which, as comparedto a corresponding subject who has not received such amount, results inimproved treatment, healing, prevention, or amelioration of a disease,disorder, or side effect, or a decrease in the rate of advancement of adisease or disorder. The term also includes within its scope amountseffective to enhance normal physiological function.

As used herein, the term “physiologically functional derivative” refersto any pharmaceutically acceptable derivative of a PPAR gamma modulator,for example, an ester or an amide, which upon administration to a mammalis capable of providing (directly or indirectly) said PPAR gammamodulator or an active metabolite thereof. Such derivatives are clear tothose skilled in the art, without undue experimentation, and withreference to the teaching of Burger's Medicinal Chemistry And DrugDiscovery, 5^(th) Edition, Vol 1: Principles and Practice, which isincorporated herein by reference to the extent that it teachesphysiologically functional derivatives.

As used herein, the term “solvate” refers to a complex of variablestoichiometry formed by a solute of a PPAR gamma modulator (or a salt orphysiologically functional derivative thereof) and a solvent. Suchsolvents for the purpose of the invention may not interfere with thebiological activity of the solute. Examples of suitable solventsinclude, but are not limited to, water, methanol, ethanol and aceticacid. Preferably the solvent used is a pharmaceutically acceptablesolvent. Examples of suitable pharmaceutically acceptable solventsinclude, without limitation, water, ethanol and acetic acid.

It will be appreciated by those skilled in the art that the PPAR gammamodulator may also be utilized in the form of a pharmaceuticallyacceptable salt or solvate thereof in the methods of the presentinvention. Suitable pharmaceutically acceptable salts can include acidor base addition salts.

A pharmaceutically acceptable acid addition salt can be formed byreaction of a compound with a suitable inorganic or organic acid (suchas hydrobromic, hydrochloric, sulfuric, nitric, phosphoric, succinic,maleic, formic, acetic, propionic, fumaric, citric, tartaric, lactic,benzoic, salicylic, glutamaic, aspartic, p-toluenesulfonic,benzenesulfonic, methanesulfonic, ethanesulfonic, naphthalenesulfonicsuch as 2-naphthalenesulfonic, or hexanoic acid), optionally in asuitable solvent such as an organic solvent, to give the salt which isusually isolated for example by crystallisation and filtration. Apharmaceutically acceptable acid addition salt can comprise or be forexample a hydrobromide, hydrochloride, sulfate, nitrate, phosphate,succinate, maleate, formate, acetate, propionate, fumarate, citrate,tartrate, lactate, benzoate, salicylate, glutamate, aspartate,p-toluenesulfonate, benzenesulfonate, methanesulfonate, ethanesulfonate,naphthalenesulfonate (e.g. 2-naphthalenesulfonate) or hexanoate salt.

A pharmaceutically acceptable base addition salt can be formed byreaction of a compound with a suitable inorganic or organic base (e.g.triethylamine, ethanolamine, triethanolamine, choline, arginine, lysineor histidine), optionally in a suitable solvent such as an organicsolvent, to give the base addition salt which is usually isolated forexample by crystallisation and filtration.

Other suitable pharmaceutically acceptable salts includepharmaceutically acceptable metal salts, for example pharmaceuticallyacceptable alkali-metal or alkaline-earth-metal salts such as sodium,potassium, calcium or magnesium salts; in particular pharmaceuticallyacceptable metal salts of one or more carboxylic acid moieties that maybe present in the compound.

Other non-pharmaceutically acceptable salts, e.g. oxalates, may be used,for example in the isolation of compounds of the invention, and areincluded within the scope of this invention.

The invention includes within its scope the use of all possiblestoichiometric and non-stoichiometric forms of the salts of thecompound.

References hereinafter to a PPAR gamma modulator include both compoundsand their pharmaceutically acceptable salts and solvates.

The PPAR gamma modulators and their pharmaceutically acceptable saltsand solvates are conveniently administered in the form of pharmaceuticalcompositions. Such compositions may conveniently be presented for use inconventional manner in admixture with one or more physiologicallyacceptable carriers or excipients. The carrier(s) must be “acceptable”in the sense of being compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

Pharmaceutical compositions may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual), rectal, nasal, topical (including buccal, sublingual ortransdermal), vaginal or parenteral (including subcutaneous,intramuscular, intravenous or intradermal) route. Such compositions maybe prepared by any method known in the art of pharmacy, for example bybringing into association the active ingredient with the carrier(s) orexcipient(s).

Pharmaceutical compositions adapted for oral administration may bepresented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Powders are prepared by comminuting thecompound to a suitable fine size and mixing with a similarly comminutedpharmaceutical carrier such as an edible carbohydrate, as, for example,starch or mannitol. Flavoring, preservative, dispersing and coloringagent can also be present.

Capsules are made by preparing a powder mixture, as described above, andfilling formed gelatin sheaths. Glidants and lubricants such ascolloidal silica, talc, magnesium stearate, calcium stearate or solidpolyethylene glycol can be added to the powder mixture before thefilling operation. A disintegrating or solubilizing agent such asagar-agar, calcium carbonate or sodium carbonate can also be added toimprove the availability of the medicament when the capsule is ingested.

Moreover, when desired or necessary, suitable binders, lubricants,disintegrating agents and coloring agents can also be incorporated intothe mixture. Suitable binders include starch, gelatin, natural sugarssuch as glucose or beta-lactose, corn sweeteners, natural and syntheticgums such as acacia, tragacanth or sodium alginate,carboxymethylcellulose, polyethylene glycol, waxes and the like.Lubricants used in these dosage forms include sodium oleate, sodiumstearate, magnesium stearate, sodium benzoate, sodium acetate, sodiumchloride and the like. Disintegrators include, without limitation,starch, methyl cellulose, agar, bentonite, xanthan gum and the like.Tablets are formulated, for example, by preparing a powder mixture,granulating or slugging, adding a lubricant and disintegrant andpressing into tablets. A powder mixture is prepared by mixing thecompound, suitably comminuted, with a diluent or base as describedabove, and optionally, with a binder such as carboxymethylcellulose, analiginate, gelatin, or polyvinyl pyrrolidone, a solution retardant suchas paraffin, a resorption accelerator such as a quaternary salt and/oran absorption agent such as bentonite, kaolin or dicalcium phosphate.The powder mixture can be granulated by wetting with a binder such assyrup, starch paste, acadia mucilage or solutions of cellulosic orpolymeric materials and forcing through a screen. As an alternative togranulating, the powder mixture can be run through the tablet machineand the result is imperfectly formed slugs broken into granules. Thegranules can be lubricated to prevent sticking to the tablet formingdies by means of the addition of stearic acid, a stearate salt, talc ormineral oil. The lubricated mixture is then compressed into tablets. Thecompounds of the present invention can also be combined with a freeflowing inert carrier and compressed into tablets directly without goingthrough the granulating or slugging steps. A clear or opaque protectivecoating consisting of a sealing coat of shellac, a coating of sugar orpolymeric material and a polish coating of wax can be provided.Dyestuffs can be added to these coatings to distinguish different unitdosages.

Oral fluids such as solution, syrups and elixirs can be prepared indosage unit form so that a given quantity contains a predeterminedamount of the compound. Syrups can be prepared by dissolving thecompound in a suitably flavored aqueous solution, while elixirs areprepared through the use of a non-toxic alcoholic vehicle. Suspensionscan be formulated by dispersing the compound in a non-toxic vehicle.Solubilizers and emulsifiers such as ethoxylated isostearyl alcohols andpolyoxy ethylene sorbitol ethers, preservatives, flavor additive such aspeppermint oil or natural sweeteners or saccharin or other artificialsweeteners, and the like can also be added.

Where appropriate, dosage unit compositions for oral administration canbe microencapsulated. The composition can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of formula (I), and salts, solvates and physiologicalfunctional derivatives thereof, can also be administered in the form ofliposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles and multilamellar vesicles. Liposomes can be formedfrom a variety of phospholipids, such as cholesterol, stearylamine orphosphatidylcholines.

The compounds of formula (I) and salts and solvates thereof may also bedelivered by the use of monoclonal antibodies as individual carriers towhich the compound molecules are coupled. The compounds may also becoupled with soluble polymers as targetable drug carriers. Such polymerscan include polyvinylpyrrolidone, pyran copolymer,polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds may becoupled to a class of biodegradable polymers useful in achievingcontrolled release of a drug, for example, polylactic acid, polepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the recipient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research, 3(6),318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions are preferably applied as a topical ointmentor cream. When formulated in an ointment, the active ingredient may beemployed with either a paraffinic or a water-miscible ointment base.Alternatively, the active ingredient may be formulated in a cream withan oil-in-water cream base or a water-in-oil base.

Pharmaceutical compositions adapted for topical administrations to theeye include eye drops wherein the active ingredient is dissolved orsuspended in a suitable carrier, especially an aqueous solvent.

Pharmaceutical compositions adapted for topical administration in themouth include lozenges, pastilles and mouth washes.

Pharmaceutical compositions adapted for rectal administration may bepresented as suppositories or as enemas.

Pharmaceutical compositions adapted for nasal administration wherein thecarrier is a solid include a coarse powder having a particle size forexample in the range 20 to 500 microns which is administered in themanner in which snuff is taken, i.e. by rapid inhalation through thenasal passage from a container of the powder held close up to the nose.Suitable compositions wherein the carrier is a liquid, foradministration as a nasal spray or as nasal drops, include aqueous oroil solutions of the active ingredient.

Pharmaceutical compositions adapted for administration by inhalationinclude fine particle dusts or mists, which may be generated by means ofvarious types of metered, dose pressurised aerosols, nebulizers orinsufflators.

Pharmaceutical compositions adapted for vaginal administration may bepresented as pessaries, tampons, creams, gels, pastes, foams or spraycompositions.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe composition isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

It should be understood that in addition to the ingredients particularlymentioned above, the compositions may include other agents conventionalin the art having regard to the type of composition in question, forexample those suitable for oral administration may include flavouringagents.

A therapeutically effective amount of a compound of the presentinvention will depend upon a number of factors including, for example,the age and weight of the animal, the precise condition requiringtreatment and its severity, the nature of the composition, and the routeof administration, and will ultimately be at the discretion of theattendant physician or veterinarian. However, an effective amount of acompound of formula (I) for the treatment of renal diseases orconditions associated with abnormal ion flux, including ADPKD, willgenerally be in the range of 0.1 to 100 mg/kg body weight of recipient(mammal) per day and more usually in the range of 1 to 10 mg/kg bodyweight per day. Thus, for a 70 kg adult mammal, the actual amount perday would usually be from 70 to 700 mg and this amount may be given in asingle dose per day or more usually in a number (such as two, three,four, five or six) of sub-doses per day such that the total daily doseis the same. When the PPAR modulator is farglitazar, dosages of 2 to 10mg per day are envisaged. An effective amount of a salt or solvate, maybe determined as a proportion of the effective amount of the compoundper se. Pharmaceutical compositions may be presented in unit dose formscontaining a predetermined amount of active ingredient per unit dose.Conveniently, unit dosage compositions are those containing a daily doseor sub dose or an appropriate fractions thereof of the activeingredient.

The PPAR gamma modulator for use in the instant invention and salts andsolvates thereof may be used in combination with one or more othertherapeutic agents. The invention thus provides in a further aspect theuse of a combination comprising a PPAR gamma modulator and salts andsolvates thereof with a further therapeutic agent or agents for use inthe prevention or treatment of renal diseases or conditions associatedwith abnormal ion flux.

When the PPAR gamma modulators are used in combination with othertherapeutic agents, the compounds may be administered eithersequentially or simultaneously by any convenient route.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above optimallytogether with a pharmaceutically acceptable carrier or excipientcomprise a further aspect of the invention. The individual components ofsuch combinations may be administered either sequentially orsimultaneously in separate or combined pharmaceutical compositions.

When combined in the same composition it will be appreciated that thetwo compounds must be stable and compatible with each other and theother components of the composition. When formulated separately they maybe provided in any convenient composition, conveniently in such a manneras are known for such compounds in the art.

When a PPAR gamma modulator is used in combination with a secondtherapeutic agent active against the same disease, the dose of eachcompound may differ from that when the compound is used alone.Appropriate doses will be readily appreciated by those skilled in theart.

The following examples are set forth to illustrate the presentinvention. Accordingly, the following Example section is in no wayintended to limit the scope of the invention contemplated herein.

EXAMPLES

In the kidney, the specific portions of the nephron in which PPAR gammais expressed include glomerular mesangial cells, renal inner medullarycollecting duct cells and renal medullary interstitial cells (Yang etal, 1999, Am. J. Physiol. 277, F966-F973). Studying the direct renalfunctional effects of these agents, in the absence of the systemiceffects on blood pressure and vascular resistance, requires an in vitrosystem with the characteristics and hormonal responsiveness of theprinciple cells of the distal nephron. Na⁺ and fluid balance areregulated by the principal cell type of the distal convoluted tubule andcortical collecting duct under the control of numerous steroid andpeptide hormones including aldosterone, insulin, vasopressin, andinsulin-like growth factor.

The Madin-Darby canine kidney (MDCK) cell line expresses manycharacteristics of the renal collecting duct. The MDCK-C7 subclone formsa high-resistance, hormone-responsive model of the principal cells,which are found in distal sections of the renal tubule. Theelectrophysiological technique of short-circuit current measurement hasbeen used to examine the response to antidiuretic hormone (ADH) in theMDCK-C7 clone. Three temporally discrete electrogenic ion transportphenomena have been described and characterized in these cells(Blazer-Yost et al, 1996; Lahr et al, 2000). Initially, the cellsexhibit anion secretion through the cystic fibrosis transmembraneconductance regulator (CFTR). The presence of CFTR was confirmed byimmunoprecipitation followed by Western blotting. The CFTR-mediatedanion secretion is transient and is followed, in time, by a verapamil-and Ba²⁺-sensitive anion secretion or cation absorption and, finally, byNa⁺ reabsorption via epithelial Na⁺ channels (ENaC). Thecharacterization of the various ion transport phenomena substantiatesthis cell line as a model renal epithelium that can be used to study thehormonal and metabolic regulation of ion transport as well as theability of pharmacological agents to affect ion transport.

Materials

The hormones and inhibitors used in these experiments were: ADH([arg]-vasopressin, Sigma, St. Louis, Mo.); amiloride hydrochloride(Sigma); NPPB (Biomol Research, Plymouth Meeting, Pa.), Verapamilhydrochloride (ICN Biomedical, Aurora, Ohio). GI262570X (Farglitazar)was provided by GlaxoSmithKline Medicinal Chemistry and may be preparedas described in U.S. Pat. No. 6,294,580.

Methods MDCK-C7 Cell Culture:

The MDCK-C7 cells were grown in a 37° C. humidified incubator with a 5%CO2 and 95% O2 gas mixture. Initially the cells were grown in 75 cm²flasks and fed with Minimal Media with Earle's salts, non-essentialamino acids and L-glutamine (MEM; Gibco/BRL, Grand Island, N.Y., USA)supplemented with 10% fetal bovine serum (Sigma), 26 mM NaHCO₃ andadjusted to pH 7.0. The confluent cells were subcultured bytrypsinization and the cells were seeded (5.4×10⁴ cell/cm²) ontoNucleopore polycarbonate membranes forming the bottom of Transwellchambers (Costar, Cambridge, Mass., USA). The Transwell chambers wereplaced in specially designed tissue culture plates to form a twocompartment system in which media are added to both apical andbasolateral surfaces. The media were aspirated and replaced three timesper week. The cells were used between passages 73 and 96.

Experimental Protocols Electrophysiological Studies:

Nucleopore filters (4.7 cm²) containing confluent MDCK-C7 cells (10-16days) were removed from the Transwell chambers and clamped between thehalves of an Ussing chamber (World Precision Instruments, Sarasota,Flo). Each half of the chamber contained a tapered fluid compartmentwith openings for voltage electrodes (close to the epithelial membrane)and current electrodes (at the opposite end of the chamber). The fluidchamber was water jacketed to maintain constant temperature (37° C.).The cells were bathed in serum-free MEM. The media were circulated inthe chambers by means of a 5% CO₂/O₂ gas lift. The electrodes wereconnected to a voltage-clamp amplifier (Current Voltage Clamp; WorldPrecision Instruments) for measurement of net ion flux as monitoredunder short-circuit conditions (SCC; short circuit current) [Ussing,1951]. Transepithelial resistance was calculated by applying a 2-mVpulse across the epithelium and measuring the resultant deflection inSCC. Data from cultures were used only if they maintained aresistance >1000 W cm². Transepithelial resistance (an indication ofcellular viability) was monitored throughout the entire duration of eachelectrophysiological experiment by pulsing the tissues with a 2000 μVpulse every 200 seconds. Resistance values were calculated from theresulting current deflections using Ohm's law.

The cultures were placed in the Ussing chambers and incubated undershort-circuited conditions until a steady baseline transport wasachieved (0.5-1 h). In the experiments to characterize the ADH-inducedion transport phenomena, the cultures were pre-incubated with theinhibitors 30 minutes prior to the addition of ADH. In the experimentswith GI262570X, compound was added 18 hrs. prior to the addition of ADH.ADH was added to the serosal bathing media; amiloride was added to theapical media 30 min after ADH to determine the portion of the transportdue to flux through the amiloride-sensitive Na⁺ channel. Theconcentrations of effectors used were: ADH, 0.1 IU/ml; amiloride, 30 μM;NPPB, 500 μM; verapamil, 25 μM, and GI262570X (concentration range from1 nM-1 μM). Each experiment was performed using matched cultures grownin parallel. The data are presented as means±SE with n indicating thenumber of different experiments.

Results

The measurement of short circuit current can be used to assess iontransport across monolayers of renal cells in culture. By convention, apositive deflection indicates cation movement from the apical to serosalbathing medium (reabsorption) or anion movement from serosal to apicalmembrane (secretion). Stimulation of MDCK-C7 cells with[arg]-vasopressin (ADH, 100 mU/ml) results in a multiphasic response inwhich there is a rapid increase in short circuit current (FIGS. 1A andB) composed of both an immediate peak of ion transport within 1 minute(FIG. 1B) followed by a more sustained maximum response over the next2-5 minutes and a later sustained response which subsequently declinestoward a pre-treatment baseline over the next 30 minutes. The latersustained responses have been shown to represent transport through theepithelial Na⁺ channel (ENaC). However, amiloride only blocks the latersustained SCC response suggesting that the earlier response to ADHrepresented events mediated by other transporters.

The identity of the ion channels and transporters involved in thecomplex response produced by ADH has been characterizedpharmacologically through the use of inhibitors as shown in FIG. 2. Inpanel A, the SCC response in cells treated with ADH alone is multiphasiccompared to untreated cells which manifest only a small sustainedincrease in SCC of approximately 1 μA/cm² in the absence of ADH. Inpanel B, NPPB (500 μM), a non-specific inhibitor of the cystic fibrosistransmembrane conductance regulator CFTR was added to the apical bathingmedium and is shown to significantly diminish the rapid transient iontransport response to ADH. There is a portion of the ion flux responseto ADH that is amiloride- and NPPB-insensitive and is blocked byverapamil (Panel C), an inhibitor of L-type Ca²⁺ as well as a modulatorof the inositol-1,4,5,-triphosphate receptor thus suggesting a role forintracellular Ca²⁺ in the response. Panel D shows the effects ofamiloride to decrease the later sustained ion transport response.

The effects of GI262570X, are graphically depicted in FIGS. 3 and 4. Thecomplete 30 minutes time-course of the ADH-induced ion transportresponse as well as the baseline stabilization period is depicted inFIG. 3 and the expanded early rapid transient response over the first 4minutes is shown in FIG. 4. In these experiments the cells werepre-incubated with GI262570X for 18 hrs prior to exposure to ADH. Therewas no compound-related effect on the baseline SCC (−10 to 0 minutes).However, comparison of the magnitude of the multiphasic SCC response toin cells exposed to ADH±GI262570X (100 nM or 1 μM, n=4/dose and n=6controls) shows that the initial rapid transient response insignificantly attenuated by both 100 nM (p<0.05) and 1 μM (p<0.01).Under these experimental conditions, there were no significant effectson the other phases of the SCC response.

It was not possible to measure a SCC response at concentrations ≧10 μMbecause the cellular resistance of the monolayers decreased to <500 ohmcm². The initial rapid transient response was inhibited over theconcentration range of 1 nM-1 μM.

Thus GI262570 decreased CFTR-mediated anion secretion in response to ADHstimulation in a cell culture model of the principal cells (MDCK-C7cells).

1. A method for treating a renal disease or a condition associated withabnormal ion flux in a mammal, the method comprising the step ofadministering to the mammal a thiazolidinedione, or a salt or a solvatethereof.
 2. The method of claim 1 wherein the thiazolidinedione ispioglitazone, rosiglitazone, or a combination thereof, or a salt or asolvate thereof.
 3. The method of claim 1 wherein the disease orcondition is autosomal dominant polycystic kidney disease.
 4. The methodof claim 1 wherein the mammal is a human.
 5. A pharmaceuticalcomposition for treating a renal disease or a condition associated withabnormal ion flux in a mammal, the composition comprising atherapeutically effective amount of one or more thiazolidinediones orsalts or solvates thereof, and one or more pharmaceutically acceptablecarriers, diluents, eluents, or a combination thereof.
 6. Thecomposition of claim 5 wherein the one or more thiazolidinedionesinclude pioglitazone or rosiglitazone, or a combination thereof, or asalt or a solvate thereof.
 7. The composition of claim 5 wherein thedisease or condition is autosomal dominant polycystic kidney disease. 8.The composition of claim 5 wherein the mammal is a human.